Lescouzeres, L. | bioRxiv Science

bioRxiv2025

Therapeutic modulation of the calpastatin/calpain pathway restores calpain-mediated synaptic proteolysis and preserves motor neurons survival and function in C9orf72 ALS

A hexanucleotide repeat expansion (GGGGCC) in the C9orf72 gene is the most prevalent genetic cause of ALS, with early neuromuscular junction (NMJ) dysfunction being a key pathological feature. Current therapies provide only limited symptomatic relief, underscoring the need for targeted, mechanism-based interventions. Using a C9orf72 ALS zebrafish model (C9-miR) and patient-derived induced pluripotent stem cell (iPSC) motor neurons, we identified significant downregulation of calpastatin, the endogenous inhibitor of calpains, a calcium-dependent protease family implicated in neurodegeneration. We demonstrate that restoring calpastatin activity with a cell-permeable calpastatin-derived peptide or the small molecule, calpeptin, ameliorates locomotor deficits and NMJ dysfunction in the C9-miR zebrafish model. These interventions enhance synaptic vesicle turnover and quantal release at the NMJ while improving motor neuron excitability and synaptic integrity in iPSC-derived motor neurons. N-terminomic/TAILS mass spectrometry revealed direct calpain-mediated cleavage of synaptic proteins in motor neurons derived from C9orf72 patients. Proteolysis of novel ALS-relevant synaptic and axonal proteins is prevented by calpeptin and calpastatin peptide treatments. Our findings establish the calpastatin as a pivotal regulator of synaptic function in C9orf72-associated ALS and identify it as a promising therapeutic target, offering a novel strategy to restore synaptic transmission and potentially halt disease progression.

neuroscience↗

bioRxiv2019

Dopamine D2 receptors modulate intrinsic properties and synaptic transmission of parvalbumin interneurons in the mouse primary motor cortex

Dopamine (DA) plays a crucial role in the control of motor and higher cognitive functions such as learning, working memory and decision making. The primary motor cortex (M1), which is essential for motor control and the acquisition of motor skills, receives dopaminergic inputs in its superficial and deep layers from the midbrain. However, the precise action of DA and DA receptor subtypes on the cortical microcircuits of M1 remains poorly understood. The aim of this work was to investigate how DA, through the activation of D2 receptors (D2R), modulates the cellular and synaptic activity of M1 parvalbumin-expressing interneurons (PVINs) which are crucial to regulate the spike output of pyramidal neurons (PNs). By combining immunofluorescence, ex vivo electrophysiology, pharmacology and optogenetics approaches, we show that D2R activation increases neuronal excitability of PVINs and GABAergic synaptic transmission between PVINs and PNs in layer V of M1. Our data reveal a mechanism through which cortical DA modulates M1 microcircuitry and might participate in the acquisition of motor skills.\n\nSignificance StatementPrimary motor cortex (M1), which is a region essential for motor control and the acquisition of motor skills, receives dopaminergic inputs from the midbrain. However, precise action of dopamine and its receptor subtypes on specific cell types in M1 remained poorly understood. Here, we demonstrate in M1 that dopamine D2 receptors (D2R) are present in parvalbumin interneurons (PVINs) and their activation increases the excitability of the PVINs, which are crucial to regulate the spike output of pyramidal neurons (PNs). Moreover the activation of the D2R facilitates the GABAergic synaptic transmission of those PVINs on layer V PNs. These results highlight how cortical dopamine modulates the functioning of M1 microcircuit which activity is disturbed in hypo- and hyperdopaminergic states.

neuroscience↗